Hepatitis B virus (HBV) infection is the most common cause of chronic liver disease on a worldwide basis and closely associated with the subsequent development of hepatocellular carcinoma (HCC). We and others have detected by monoclonal antibodies and molecular techniques HBV and/or variants in individuals with chronic liver disease and hepatocellular carcinoma previous thought to be unrelated to HBV infection. We wish to characterize these viral agents in more detail at the molecular and antigenic level, to investigate the biochemical basis of their variant biological behavior, and to evaluate their significance in the pathogenesis of HCC. We plan to do the following: 1) Evaluate the clinical and epidemiological significance of infection with low level HBV and/or variants and to assess the contribution of hepatitis C viral (HCV) infection. We will employ the polymerase chain reaction to detect, amplify and characterize low level viral sequences in serum and liver of HBsAg-negative patients with hepatocellular carcinoma. In this regard, our aims are as follows: a. Identify such agents by a rapid, sensitive and specific technique using monoclonal anti-HBs antibody to capture encapsidated virions in serum following by PCR amplification of different regions of the HBV genome. b. Use a restriction endonuclease fragment analysis following MAb capture/PCR to rapidly characterize the HBV related agents. This method will allow us to rapidly assess genomic heterogeneity. c. Demonstrate the presence of low level HBV DNA in liver of these patients and compare our results to findings in serum. d. Attempt to determine the prevalence of HCV infection by measuring the presence of antibody to HCV and HCV genome using recently available anti-HCV diagnostic kit and PCR technique, respectively. 2) Clone and sequence HBV DNA from HBsAg-negative patients in order to determine the nucleotide and amino acid variability which may be the molecular basis for their biological behavior. We also plan to explore the role of viral factor(s) in the pathogenesis of liver disease. 3) Examine the physiologic significance of genomic mutations in HBV variants in vitro in order to understand their biological properties in hepatocellular carcinoma. We will reconstruct and transfect the variant HBV genomes into human hepatoma cell lines and primary hepatocyte cultures and analyze viral antigen production and composition, transcriptional regulation, and replication competence of these variants. Based on our preliminary data obtained, we are optimistic that new information will be obtained on the clinical significance and molecular characteristics of HBV genetic variants. We believe that these agents may play a previously unrecognized role in the pathogenesis of chronic liver disease leading to or contributing to HCC. We also believe that by studying these variants in vitro and in vivo, further insights into the biological significance of genetic mutants of HBV will be obtained.